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Cloning and functional characterization of two cDNAs encoding NADPH-dependent 3-ketoacyl-CoA reductased from developing cotton fibers

摘要Genes encoding enzymes involved in biosynthesis of very long chain fatty acids were significantly up-regulated during early cotton fiber development. Two cDNAs, GhKCR1 and GhKCR2 encoding putative cotton 3-ketoacyl-CoA reductases that catalyze the second step in fatty acid elongation, were isolated from developing cotton fibers. GhKCR1 and 2 contain open reading frames of 963 bp and 924 bp encoding proteins of 320 and 307 amino acid residues,respectively. Quantatitive RT-PCR analysis showed that both these genes were highly preferentially expressed during the cotton fiber elongation period with much lower levels recovered from roots, stems and leaves. GhKCR1 and 2 showed 30%-32% identity to Saccharomyces cerevisiae Ybr159p at the deduced amino acid level. These cotton cDNAs were cloned and expressed in yeast haploid ybr159w△ mutant that was deficient in 3-ketoacyl-CoA reductase activity.Wild-type growth rate was restored in ybr159w△ cells that expressed either GhKCR1 or 2. Further analysis showed that GhKCR1 and 2 were co-sedimented within the membranous pellet fraction after high-speed centrifugation, similar to the yeast endoplasmic reticulum marker ScKar2p. Both GhKCR(s) showed NADPH-dependent 3-ketoacyl-CoA reductase activity in an in vitro assay system using palmitoyl-CoA and malonyl-CoA as substrates. Our results suggest that GhKCR1 and 2 are functional orthologues of ScYbr159p.

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作者单位 National Laboratory of Protein Engineering and Plant Genetic Engineering, College of Life Sciences, Peking University,Beijing, 100871, China;Department of Biochemistry and Molecular Biology, College of Life Sciences, Peking University, Beijing, 100871, China [1] Biocenter Oulu and Department of Biochemistry, University of Oulu, P.O. Box 3000, FIN-90014 University of Oulu, Finland [2]
分类号 R3
DOI 10.1038/sj.cr.7290315
发布时间 2005-08-18
基金项目
国家重点基础研究发展计划(973计划) 国家自然科学基金 Academy of Finland Fund
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细胞研究(英文版)

细胞研究(英文版)

2005年15卷6期

465-473页

SCIMEDLINEISTICCSCDBP

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